The invention is generally in the field of transgenic animals and more specifically in the field of transgenic animal models of prostate cancer.
The prostate is derived from the urogenital sinus. Cross sections through this tubuloalveolar gland reveal a simple cuboidal or columnar epithelium, with no apparent differences between the cells lining its ducts and those lining its acini (reviewed in Lepor and Lawson, eds., "Prostatic Diseases" (W. B. Saunders Company, Philadelphia, 1993). The epithelium contains three cell types. Secretory luminal cells are the predominant cell type. Neuroendocrine cells are scattered between luminal cells. Basal cells lie between luminal cells and the basement membrane (reviewed in Lepor and Lawson (1993), and Bonkhoff, Eur. Urol. 30:201-205 (1996)).
The prostatic epithelium undergoes continuous self-renewal. However, turnover is on the order of months, so dividing cells are rarely observed unless there is tissue damage. Surprisingly little is known about the regulation of proliferation and differentiation in the normal mouse or human prostate. Basal cells comprise greater than 70% of the proliferating cell population in the normal adult prostate. Intermediate cell types that share features of more than one of the three lineages have been identified in normal and in neoplastic prostatic epithelium. This has led to speculation that the three lineages are interrelated and possibly derived from a common progenitor (Bonkhoff and Remberger, Prostate 28:98-106 (1996); Bonkhoff (1996)). There is some indirect evidence that this progenitor may reside within the basal cell population (reviewed in Bonkhoff (1996)).
After puberty, the prostatic epithelium requires androgens for maintenance of its proliferation and for prevention of cell death (Isaacs et al., Seminars in Cancer Biol. 5:391-400 (1994)). If the normal prostate is deprived of androgens after puberty, it will involute. The prostate never completes its morphogenesis in dogs or rats if they are castrated before sexual maturity. If these animals are supplied with testosterone later in life, the prostate can complete its maturation--suggesting the existence of a stem cell that does not depend upon androgens for its survival (reviewed in Lepor and Lawson (1993)).
Adenocarcinoma of the prostate is the most frequently diagnosed cancer in men in the United States, and is the second leading cause of male cancer deaths (Karp et al., Cancer Res. 56:5547-5556 (1996)). The exquisite susceptibility of this organ to cancer in humans is not understood. Skenes glands represent the female homolog of the prostate but are not a site where neoplastic transformation is observed.
Increasing numbers of patients with occult prostate cancer are being identified by a rapid, sensitive screening assay for prostate specific antigen (PSA). Large scale PSA screening has produced a dilemma, since most patients with carcinoma in situ will die of other causes before suffering symptoms of their cancer. Those individuals whose disease will progress rapidly may be saved if they are treated early. However, these individuals are not easily distinguished from those who will only have indolent disease (Lepor and Lawson (1993), and Karp et al. (1996)). A better understanding of the molecular mechanisms leading to malignant transformation of the prostate is needed to explain its high prevalence, and to assist in the identification of those patients who are at risk for aggressive disease and who, therefore, would benefit from aggressive therapy.
Surgical or chemical androgen ablation is often used to treat patients when their adenocarcinoma is no longer confined to the prostate. This approach is effective initially in 60 to 80% of patients. However, the cancers inevitably become androgen-independent (reviewed in Lepor and Lawson (1993)). This may be due to selection of a small number of possibly pre-existing androgen-independent cells, or to conversion of androgen-dependent cells to an androgen-independent state (Isaacs and Coffey, Cancer Res. 41:5070-5075 (1981); Rennie et al., J. Steroid Biochem. Mol. Biol. 37:843-847 (1990)). Mutations in the androgen receptor have been identified in some androgen-independent human prostatic tumors. However, the presence or absence of the androgen receptor does not correlate with the androgen-dependence of prostatic cancers in the same the way that the presence or absence of estrogen and progesterone receptors correlate with the hormone-dependence of breast cancers (reviewed in Sluyser, Crit. Rev. Onc. 5:539-554 (1994)). Therefore, it is difficult to predict whether an individual patient will respond to androgen ablation therapy. A better understanding of the evolution of androgen resistance is important for the development of better treatment strategies for human prostate cancer.
Several observations suggest that the anti-apoptotic regulator Bcl-2 plays a role in development of androgen-independence. Bcl-2 is normally expressed in basal cells, which do not show the same apoptotic response to androgen withdrawal as luminal cells (Rouleau et al., Mol. Endocrinol. 4:2003-2013 (1990)). Prostate cancer cells expressing Bcl-2 in vitro and in vivo are also resistant to androgen withdrawal (Raffo et al., Cancer Res. 55:4438-4445 (1995)). Colombel et al., Am. J. Path. 143:390-400 (1993), conducted a retrospective study of human prostatic adenocarcinomas. They included primary lesions as well as metastatic foci in their survey. Their analysis indicated that all androgen-independent tumors from patients with hormone-refractory disease produce Bcl-2. In contrast, only a subset of androgen-dependent tumors contained detectable levels of Bcl-2. Similar results were obtained in a separate study (McDonnell et al., Cancer Res. 52:6940-6944 (1992)). Based on these results, Colombel and coworkers hypothesized that Bcl-2 expression might enable prostatic cancer cells to survive in an androgen-deprived environment and confer resistance to androgen withdrawal therapies. This hypothesis needs to be tested in a genetically well defined model of human prostatic cancer.
Fifty to seventy five percent of prostatic adenocarcinomas show some neuroendocrine differentiation, as defined by immunohistochemical stains for markers such as chromogranin A. Although the significance of this phenotype is controversial, some data indicate that neuroendocrine differentiation correlates with a hormone refractory state and a poor long term prognosis (reviewed in Logothetis and Hoosein, "Comprehensive Textbook of Genitourinary Oncology" (Williams & Wilkins, Baltimore, 1996); di Sant'Agnese and Cockett, Cancer 78:357-361 (1996)). Two studies have demonstrated expression of Bcl-2 in prostatic secretary (luminal) cells that are in contact with neuroendocrine cells (Cohen et al., Pathology 27:229-232 (1995); Segal et al., Arch. Pathol. Lab Med. 118:616-618 (1994)). Bcl-2 is not normally detected in luminal cells. These observations have led to the notion that signalling between neuroendocrine cells and luminal cells results in induction of Bcl-2 in luminal cells. Since the presence of neuroendocrine cells in adenocarcinoma may correlate with an androgen-independent state, it is possible that this induction of Bcl-2 expression by neuroendocrine cells may be related to development of androgen-independence.
Finally, prostatic intraepithelial neoplasia (PIN) is generally believed to be a precursor to adenocarcinoma of the prostate although formal proof is lacking. A genetically well-defined and manipulatable model is needed that can be used to dissect the molecular events associated with development of, and progression from, PIN.
Three transgenic models of prostate cancer have been reported in the literature. Each model has used different transcriptional regulatory elements to drive expression of simian virus 40 large T antigen (TAg).
The human fetal G.gamma. globin promoter was used to express TAg in erythroid cells (Perez-Stable et al., Lab Invest. 74:363-373 (1996); Perez-Stable et al., Cancer Res. 57:900-906 (1997)). In one G.gamma.-TAg pedigree, males developed prostate tumors that showed "mixed neuroendocrine and epithelial cell features". Females developed adrenocortical tumors. Prostatic neoplasia appeared relatively late. PIN was observed in only a subset of transgenic males by 4 to 5 months of age. However, by 6 months, 90% of hemizygous males had palpable tumor masses. Tumors were said to visibly "metastasize" to lymph nodes, adrenal glands, and the kidney but the issue of metastastic spread is confounded by the fact that the transgene is expressed in some of these organs in this and other pedigrees. This model is the only one of the three published models of prostatic cancer in which neuroendocrine differentiation is reported. Several neuroendocrine cell markers were detected by Western blot but the presence of secretory granules in tumor cells could not be confirmed by EM. Based on these findings, the authors postulated that TAg was expressed in a luminal epithelial cell with neuroendocrine features. When castration was performed after puberty, tumor development was unaffected, suggesting androgen-independence. The fact that prostatic cancer was only seen in one of several lines of G.gamma.-TAg transgenic mice suggests an insertion site effect.
Maroulakou et al., Proc. Natl. Acad. Sci. USA 91:11236-11240 (1994) and Shibata et al., Cancer Res. 56:4894-4903 (1996) reported that several pedigrees of mice expressing TAg under the control of the rat prostatic steroid binding protein promoter develop incompletely penetrant prostate tumors. TAg is initially expressed in "scattered apparently normal-appearing prostate epithelial cells" (Maroulakou et al. (1994)). PIN does not appear in some animals until 8 months of age. Prostate tumors often do not develop for 6 to 8 months and are not metastatic. The authors do not describe any experiments that tested the androgen-sensitivity of these lesions. Furthermore, these mice have significant health problems due to transgene expression at other sites: that is, bone, cartilage, thyroid, salivary glands and the nasal epithelium.
The rat probasin gene (rPB) encodes an androgen- and zinc-regulated protein that is only expressed in the dorsolateral prostatic epithelium. Greenberg et al. Proc. Natl. Acad. Sci. USA 92:3439-3443 (1995) and Gingrich et al., Cancer Res. 56:4096-4102 (1996) found that nucleotides -426 to +28 of rPB could be used to express foreign gene products to this cell population in transgenic mice. Probasin-TAg transgenic mice develop androgen-dependent prostate cancer. However, there is significant variability in the rate of tumor development between transgenic lines, attributed to varying levels of TAg. For example, members of one high-expressing pedigree develop large, multinodular tumors by 10 weeks of age, whereas members of another, low-expressing pedigree show only profound hyperplasia at 33 weeks. Expression of TAg is directed to mature luminal epithelial cells, where the presence of the oncoprotein precedes transformation (PIN). The authors noted that "positively staining nuclei were plentiful in epithelial cells lining some of the nontumoral glands, and interestingly, most of the TAg-positive nuclei were indistinguishable from their unstained neighbors" (Gingrich et al. (1996)). Metastases occur in this model.
Accordingly, it would be useful to have an animal model of prostate cancer that provides a consistent phenotype, where neoplasia progresses more rapidly, and produces hormone refractory tumors.
Therefore, it is an object of the present invention to provide a transgenic animal model of prostate cancer.
It is another object of the present invention to provide a method of testing compounds for an effect on initiation, progression, or both, of prostate tumors.